U.S. patent application number 09/998350 was filed with the patent office on 2003-04-24 for redox-stable, non-phosphorylated cyclic peptide inhibitors of sh2 domain binding to target protein, conjugates thereof, compositions and methods of synthesis and use.
This patent application is currently assigned to The Govt. of the United States of America. Invention is credited to King, C. Richter, Long, Ya-Qiu, Lung, Feng-Di T., Roller, Peter P., Yang, Dajun.
Application Number | 20030078368 09/998350 |
Document ID | / |
Family ID | 26835002 |
Filed Date | 2003-04-24 |
United States Patent
Application |
20030078368 |
Kind Code |
A1 |
Roller, Peter P. ; et
al. |
April 24, 2003 |
Redox-stable, non-phosphorylated cyclic peptide inhibitors of SH2
domain binding to target protein, conjugates thereof, compositions
and methods of synthesis and use
Abstract
A compound of formula: 1 in which L is sulfur, sulfoxide, oxygen
or methylene, and a compound of formula: 2 in which (i) aa.sup.1 is
Adi and aa.sup.4 is Glu or (ii) each of aa.sup.1 and aa.sup.4 is
Adi, L is sulfur, sulfoxide, oxygen or methylene, which compounds
(and their conjugates) bind to an SH2 domain in a protein
comprising an SH2 domain, are non-phosphorylated, are redox-stable
in vivo, are characterized by an IC.sub.50 in vivo of less than
about 4.0 .mu.M with respect to the SH2 domain in Grb2, and, upon
binding to the SH2 domain of Grb2, have a turn conformation. A
conjugate comprising a compound as described above and a carrier
agent, a composition comprising (i) a compound or a conjugate as
described above and (ii) a carrier, a method of inhibiting binding
of an SH2 domain in a protein comprising an SH2 domain to a target
protein in an animal, wherein the SH2 domain is contacted with a
target protein-binding inhibiting effective amount of a compound or
a conjugate as described above, and a method of synthesizing such
conjugates.
Inventors: |
Roller, Peter P.;
(Rockville, MD) ; Long, Ya-Qiu; (Bethesda, MD)
; Lung, Feng-Di T.; (Taichung, TW) ; King, C.
Richter; (Washington, DC) ; Yang, Dajun;
(Rockville, MD) |
Correspondence
Address: |
LEYDIG VOIT & MAYER, LTD
TWO PRUDENTIAL PLAZA, SUITE 4900
180 NORTH STETSON AVENUE
CHICAGO
IL
60601-6780
US
|
Assignee: |
The Govt. of the United States of
America
Suite 325 6011 Executive Boulevard
Rockville
MD
20852
|
Family ID: |
26835002 |
Appl. No.: |
09/998350 |
Filed: |
November 30, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09998350 |
Nov 30, 2001 |
|
|
|
PCT/US00/15201 |
Jun 2, 2000 |
|
|
|
60137187 |
Jun 2, 1999 |
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Current U.S.
Class: |
530/317 |
Current CPC
Class: |
C07K 7/06 20130101; C07K
7/56 20130101; A61K 38/00 20130101 |
Class at
Publication: |
530/317 ;
514/9 |
International
Class: |
A61K 038/12; C07K
007/64 |
Claims
What is claimed is:
1. A compound of formula: 8in which L is sulfur, sulfoxide, oxygen
or methylene, in which, optionally, one or more of Tyr.sup.3,
Glu.sup.4, Val.sup.6, Met.sup.8 and Tyr.sup.9 is modified, and in
which, optionally, there is a conservative or neutral amino acid
substitution at either one or both of Leu.sup.2 and Gly.sup.7,
wherein said compound binds an Src homology 2 (SH2) domain in a
protein comprising an SH2 domain, is non-phosphorylated, is
redox-stable in vivo, and is characterized by an IC.sub.50 in vivo
of less than about 4.0 .mu.M when the target protein is growth
factor receptor-bound protein 2 (Grb2), and whereupon binding to
Grb2, the compound has a turn conformation.
2. The compound of claim 1, wherein said IC.sub.50 in vivo is less
than or equal to 2.0 .mu.M.
3. A compound of formula: 9in which L is sulfur, sulfoxide, oxygen
or methylene, in which (i) aa.sup.1 is Adi and aa.sup.4 is Glu or
(ii) each of aa.sup.1 and aa.sup.4 is Adi, in which, optionally,
one or more of Tyr.sup.3, Val.sup.6, Met.sup.8 and Tyr.sup.9 is
modified, and in which, optionally, there is a conservative or
neutral amino acid substitution at either one or both of Leu.sup.2
and Gly.sup.7, wherein said compound binds an SH2 domain in a
protein comprising an SH2 domain, is non-phosphorylated, is
redox-stable in vivo, and is characterized by an IC.sub.50 in vivo
of less than about 4.0 .mu.M when the target protein is Grb2, and
whereupon binding to Grb2, the compound has a turn
conformation.
4. The compound of claim 3, wherein said IC.sub.50 in vivo is less
than or equal to 2.0 .mu.M.
5. A conjugate comprising a compound of claim 1 and a carrier
agent.
6. The conjugate of claim 5, wherein said carrier agent is a signal
peptide, antennapedia peptide, or lipofectin.
7. A conjugate comprising a compound of claim 3 and a carrier
agent.
8. The conjugate of claim 7, wherein said carrier agent is a signal
peptide, antennapedia peptide, or lipofectin.
9. A composition comprising (i) a compound of claim 1 or a
conjugate comprising a compound of claim 1 and a carrier agent and
(ii) a carrier.
10. A composition comprising (i) a compound of claim 3 or a
conjugate comprising a compound of claim 3 and a carrier agent and
(ii) a carrier.
11. A method of inhibiting binding of an SH2 domain in a protein
comprising an SH2 domain to a target protein in an animal, which
method comprises contacting said SH2 domain with a target
protein-binding inhibiting amount of (i) a compound of formula 10in
which L is sulfur, sulfoxide, oxygen or methylene, in which,
optionally, one or more of Tyr.sup.3, Glu.sup.4, Val.sup.6,
Met.sup.8 and Tyr.sup.9 is modified, and in which, optionally,
there is a conservative or neutral amino acid substitution at
either one or both of Leu.sup.2 and Gly.sup.7, wherein said
compound binds an SH2 domain in a protein comprising an SH2 domain,
is non-phosphorylated, is redox-stable in vivo, and is
characterized by an IC.sub.50 in vivo of less than about 4.0 .mu.M
when the target protein is Grb2, and whereupon binding to Grb2, the
compound has a turn conformation, (ii) a compound of formula 11in
which L is sulfur, sulfoxide, oxygen or methylene, in which (i)
aa.sup.1 is Adi and aa.sup.4 is Glu or (ii) each of aa.sup.1 and
aa.sup.4 is Adi, in which, optionally, one or more of Tyr.sup.3,
Val.sup.6, Met.sup.8 and Tyr.sup.9 is modified, and in which,
optionally, there is a conservative or neutral amino acid
substitution at either one or both of Leu.sup.2 and Gly.sup.7,
wherein said compound binds an SH2 domain in a protein comprising
an SH2 domain, is non-phosphorylated, is redox-stable in vivo, and
is characterized by an IC.sub.50 in vivo of less than about 4.0
.mu.M when the target protein is Grb2, and whereupon binding to
Grb2, the compound has a turn conformation, or (iii) a conjugate
comprising either of the forgoing compounds and a carrier agent,
whereupon binding of said compound or said conjugate to said SH2
domain, binding of said SH2 domain to said target protein is
inhibited.
12. The method of claim 11, wherein said target protein is a growth
factor receptor.
13. The method of claim 11, wherein said target protein is a
morphology determining protein.
14. The method of claim 11, wherein said target protein is a
cellular attachment protein.
15. The method of claim 11, wherein said target protein is a
proto-oncoprotein or an oncoprotein.
16. The method of claim 11, wherein said target protein is
mitogen-activated protein (MAP) kinase.
17. The method of claim 1, wherein inhibition of binding of said
SH2 domain to said target protein prevents cancer.
18. The method of claim 17, wherein said cancer is breast
cancer.
19. The method of claim 18, which method further comprises
administering to said animal an effective amount of an anti-cancer
agent, wherein inhibition of binding of said SH2 domain to said
target protein and administration of an effective amount of an
anti-cancer agent treats cancer.
20. The method of claim 19, wherein said anti-cancer agent is a
cancer chemotherapeutic agent, radiation and/or a radioactive
isotope.
21. A method of synthesizing a conjugate comprising (i) a compound
of formula 12in which L is sulfur, sulfoxide, oxygen or methylene,
in which, optionally, one or more of Tyr.sup.3, Glu.sup.4,
Val.sup.6, Met.sup.8 and Tyr.sup.9 is modified, and in which,
optionally, there is a conservative or neutral amino acid
substitution at either one or both of Leu.sup.2 and Gly.sup.7,
wherein said compound binds an Src homology 2 (SH2) domain in a
protein comprising an SH2 domain, is non-phosphorylated, is
redox-stable in vivo, and is characterized by an IC.sub.50 in vivo
of less than about 4.0 .mu.M when the target protein is growth
factor receptor-bound protein 2 (Grb2), and whereupon binding to
Grb2, the compound has a turn conformation, or a compound of
formula 13in which L is sulfur, sulfoxide, oxygen or methylene, in
which (i) aa.sup.1 is Adi and aa.sup.4 is Glu or (ii) each of
aa.sup.1 and aa.sup.4 is Adi, in which, optionally, one or more of
Tyr.sup.3, Val.sup.6, Met.sup.8 and Tyr.sup.9 is modified, and in
which, optionally, there is a conservative or neutral amino acid
substitution at either one or both of Leu.sup.2 and Gly.sup.7,
wherein said compound binds an SH2 domain in a protein comprising
an SH2 domain, is non-phosphorylated, is redox-stable in vivo, and
is characterized by an IC.sub.50 in vivo of less than about 4.0
.mu.M when the target protein is Grb2, and whereupon binding to
Grb2, the compound has a turn conformation, and (ii) a carrier
agent, which method comprises: (i) synthesizing from C-terminus to
N-terminus a linear side-chain protected peptide comprising from
N-terminus to C-terminus the amino acid sequence of the compound
and the amino acid sequence of a carrier agent on an amide resin,
(ii) N-terminally haloacetylating the peptide, (iii) cleaving the
peptide from the resin and, either simultaneously or sequentially,
deprotecting the side-chains of the peptide, (iv) nucleophilically
displacing the N-terminal halo group with the cysteine side-chain
thiol functionality at from about pH 7 to about pH 8, and (v)
purifying the resulting conjugate.
22. The method of claim 21, wherein haloacetylating is
bromoacetylating or chloroacetylating.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention relates to redox-stable,
non-phosphorylated cyclic peptide inhibitors of SH2 domain binding
to a target protein and conjugates of such cyclic peptide
inhibitors, as well as compositions, methods of synthesis and
methods of use.
BACKGROUND OF THE INVENTION
[0002] Src homology 2 (SH2) domains selectively bind to
phosphotyrosyl (pTyr)-containing regions of target proteins. SH2
binding can result in the modulation of c-src activity (Cooper et
al., Cell 73: 1051-1054 (1993)), the alteration of the substrate
specificity of c-abl proto-oncoproteins (Mayer et al., Mol. Cell.
Biol. 14: 2883-2894 (1994); Feller et al., Trends Biochem. Sci. 19:
453-458 (1994)) and the transduction of signals initiated at growth
factor receptors (Margolis et al., J. Am. Soc. Nephrol. 5:
1288-1299 (1994)) and cellular attachment systems (Schlaepfer et
al., Nature 372: 786-791 (1994)). Thus, the SH2 domain has been
suggested to be a promising site for therapeutic intervention
(Brugge, Science 260: 918-919 (1993)).
[0003] The SH2 domain of growth factor receptor-bound protein 2
(Grb2), a 24 kda intracellular carrier agent that is composed of
one SH2 domain and two src homology 3 (SH3) domains, mediates
cellular signaling by binding pTyr-containing motifs within several
proteins, although the Grb2 SH2 domain also can bind to proteins
that do not contain pTyr (Oligino et al., J. Biol. Chem. 272:
29046-29052 (1997)). Such proteins include the adapter protein SHC
(via p52 binding; Pelicci et al., Cell 70: 93-104 (1992);
Rozakis-Adcock et al., Nature 360: 689-692 (1992); Sastry et al.,
Oncogene 11: 1107-1112 (1995); Janes et al., Oncogene 9: 3601-3608
(1994); Daly et al., Oncogene 9: 2723-2727 (1994)), growth factor
receptors, such as epidermal growth factor receptors (EGFR) and
their oncogenic analog receptors, the erbB receptors
(Rozakis-Adcock et al. (1992), supra; Lowenstein et al., Cell 70:
431-442 (1992); Gale et al., Nature 363: 88-92 (1993); Buday et
al., Cell 73: 611-620 (1993); Egan et al., Nature 363: 45-51
(1993)), in particular erbB2 (HER-2 or neu; via p185 binding;
Sastry et al. (1995), supra; Janes et al. (1994), supra; Daly et
al. (1994), supra; Xie et al., J. Biol. Chem. 270: 30717-30724
(1995); Gishizky et al., PNAS USA 92: 10889-10893 (1995)),
morphology-determining proteins, such as cytoplasmic focal adhesion
protein-tyrosine kinase (FAK; Schlaepfer et al. (1994), supra), and
cellular oncoproteins, such as BCR-abl (Gale et al. (1993), supra;
Pendergast et al., Cell 75: 175-185 (1993); Xie et al. (1995),
supra; Gishizky et al. (1995), supra).
[0004] The prevention of Grb2-mediated multiprotein assemblies is
considered to be a promising therapeutic target for the development
of antiproliferative agents directed to cells that over-express
growth factor receptors. In this regard, a non-phosphorylated
peptide containing II amino acids in which the side chains of the
two terminal cysteine residues form a ring has been reported to
bind selectively the SH2 domain of Grb2 (Oligino et al. (1997),
supra). The cyclic structure and the size of the ring have been
determined to be critical to the binding activity of the
non-phosphorylated peptide, which has been designated G1. An
equipotent redox-stable thioether cyclized analog, GITE, also has
been reported (Oligino et al. (1997), supra). Unfortunately, G1 and
G1 TE have receptor affinities that are substantially lower than a
segment of the naturally phosphorylated ligand, i.e.,
SHC(pY.sup.317)-9-mer (IC.sub.50 of 10-25 .mu.M and 10-15 .mu.M,
respectively, vs. 1.0 .mu.M). Accordingly, the utility of G1 and
GITE in in vivo prophylactic and therapeutic applications is
limited.
[0005] In view of the above, there remains a need for an effective
inhibitor of binding of an SH2 domain in a protein comprising an
SH2 domain to a target protein. Therefore, it is an object of the
present invention to provide non-phosphorylated cyclic peptide
inhibitors of SH2 domains to target proteins, such as the SH2
domain that exists in Grb2 protein, which are redox-stable in vivo
and are characterized by receptor affinities that are substantially
better than currently available cyclic peptide inhibitors of SH2
domains. It is another object of the present invention to provide
conjugates of such cyclic peptide inhibitors in order to facilitate
cellular internalization of active agents. It is yet another object
of the present invention to provide a composition comprising (i)
such an inhibitor or such a conjugate and (ii) a carrier. It is
still yet another object of the present invention to provide a
method of synthesizing such conjugates. A further object of the
present invention is to provide methods of use of such inhibitors
and conjugates thereof. These and other objects and advantages, as
well as additional inventive features, will be apparent from the
detailed description provided herein.
BRIEF SUMMARY OF THE INVENTION
[0006] The present invention provides a compound of formula: 3
[0007] in which L is sulfur, sulfoxide, oxygen or methylene, and,
optionally, one or more of Tyr.sup.3, Glu.sup.4, Val.sup.6,
Met.sup.8 and Tyr.sup.9 is modified. The present invention also
provides a compound of formula: 4
[0008] in which (i) aa.sup.1 is Adi and aa.sup.4 is Glu or (ii)
each of aa.sup.1 and aa.sup.4 is Adi, and in which, L is sulfur,
sulfoxide, oxygen or methylene, and, optionally, one or more of
Tyr.sup.3, Val.sup.6, Met.sup.8 and Tyr.sup.9 is modified. With
respect to these compounds, the modifications of the indicated
amino acids desirably do not result in a highly charged moiety that
prevents the compound from entering into a cell. Optionally, there
is a conservative or neutral amino acid substitution at either one
or both of Leu.sup.2 and Gly.sup.7. The compounds (and their
conjugates) bind an SH2 domain in a protein comprising an SH2
domain, are non-phosphorylated, are redox-stable in vivo, and are
characterized by an IC.sub.50 in vivo of less than about 4.0 .mu.M
with respect to the SH2 domain in Grb2, and, upon binding to the
SH2 domain of Grb2, the compounds have a turn conformation.
Preferably, the compounds (and their conjugates) have an IC.sub.50
in vivo of less than or equal to 2.0 .mu.M.
[0009] Also provided by the present invention is a conjugate
comprising a compound as described above and a carrier agent that
facilitates cellular internalization. In addition, the present
invention provides a composition comprising a compound or a
conjugate as described above and a carrier.
[0010] A method of inhibiting binding of an SH2 domain in a protein
comprising an SH2 domain to a target protein in an animal is
further provided. The method comprises contacting the SH2 domain in
a protein comprising an SH2 domain with a target protein-binding
inhibiting amount of a compound or a conjugate as described above,
whereupon binding of the SH2 domain in the protein comprising the
SH2 domain to the target protein is inhibited.
[0011] Still further provided is a method of synthesizing a
conjugate described above. The method comprises:
[0012] (i) synthesizing from C-terminus to N-terminus a linear
side-chain protected peptide comprising from N-terminus to
C-terminus the amino acid sequence of the compound of claim 1 or 2
and the amino acid sequence of a carrier agent on an amide
resin,
[0013] (ii) N-terminally haloacetylating the peptide,
[0014] (iii) cleaving the peptide from the resin and, either
simultaneously or sequentially, deprotecting the side-chains of the
peptide,
[0015] (iv) nucleophilically displacing the N-terminal halo group
with the cysteine side-chain thiol functionality at from about pH 7
to about pH 8, and
[0016] (v) purifying the resulting conjugate.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention is predicated on the surprising and
unexpected discovery of non-phosphorylated cyclic peptide
inhibitors of binding of SH2 domains in proteins comprising SH2
domains to target proteins which not only are redox-stable in vivo
but have unprecedented specific binding affinities. Previously
reported non-phosphorylated cyclic peptide inhibitors of binding of
SH2 domains in proteins comprising SH2 domains to target proteins
had specific binding affinities that were substantially worse than
the naturally occurring ligand of SH2 domains, rendering them
unsuitable for in vivo applications. The discovery of such
redox-stable, non-phosphorylated cyclic peptide inhibitors has
involved the painstaking analysis of amino acid sequences, size
constraints, the presence and absence of phosphorylation, such as
that which occurs in the naturally occurring ligand of SH2 domains,
cyclization linkages, modifications of amino acid side chains,
affinity binding studies and molecular modeling.
[0018] In view of the above, the present invention generally
provides a compound of formula: 5
[0019] in which Xxx.sup.1 represents a standard, naturally
occurring or non-natural amino acid other than glutamic acid and L
is sulfur, sulfoxide, oxygen or methylene. The side chain of
Xxx.sup.1 can be extended by one or more methylene groups provided
that the compound can bind to an SH2 domain in a protein comprising
an SH2 domain in accordance with the present invention. An
intrachain methylene of the side chain can be substituted with O, S
or N. In addition, the side chain can be terminally substituted
with one or more of F and/or COOH. The other three-letter
designations represent standard, naturally occurring and
non-natural amino acids. The compound (and its conjugate) binds an
SH2 domain in a protein comprising an SH2 domain, is
non-phosphorylated, is redox-stable in vivo, and is characterized
by an IC.sub.50 in vivo of less than about 4.0 .mu.M with respect
to the SH2 domain in Grb2. Preferably, the compound (and its
conjugate) has an IC.sub.50 in vivo of less than or equal to 2.0
.mu.M. Upon binding to the SH2 domain of Grb2, the compound has a
turn conformation. Optionally, one or more of Tyr.sup.3, Glu.sup.4,
Val.sup.6, Met.sup.8 and Tyr.sup.9 is modified; desirably, the
modification does not result in a highly charged moiety that
prevents the compound from entering into a cell. Also optionally,
there is a conservative or neutral amino acid substitution at
either one or both of Leu.sup.2 and Gly.sup.7.
[0020] By "modified" is meant substitution by a naturally occurring
amino acid, a D-configuration analog thereof or a non-natural amino
acid. Whether or not a compound is redox-stable in vivo can be
determined in accordance with methods known in the art.
[0021] Specifically, the present invention provides a compound of
formula: 6
[0022] in which Gla represents .gamma.-carboxy-L-glutamic acid or a
closely related methylene (CH.sub.2).sub.x side chain-extended
analog in which x is 2 or 3, the other three-letter designations
represent standard, naturally occurring and non-natural amino acids
and L is sulfur, sulfoxide, oxygen or methylene. The compound (and
its conjugate) binds an SH2 domain in a protein comprising an SH2
domain, is non-phosphorylated, is redox-stable in vivo, and is
characterized by an IC.sub.50 in vivo of less than about 4.0 .mu.M
with respect to the SH2 domain in Grb2. Preferably, the compound
(and its conjugate) has an IC.sub.50 in vivo of less than or equal
to 2.0 AM. Upon binding to the SH2 domain of Grb2, the compound has
a turn conformation. Optionally, one or more of Tyr.sup.3,
Glu.sup.4, Val.sup.6, Met.sup.8 and Tyr.sup.9 is modified;
desirably, the modification does not result in a highly charged
moiety that prevents the compound from entering into a cell. Also
optionally, there is a conservative or neutral amino acid
substitution at either one or both of Leu.sup.2 and Gly.sup.7.
[0023] The present invention further provides a compound of
formula: 7
[0024] in which aa represents an amino acid as indicated, the other
three-letter designations represent standard, naturally occurring
and normatural amino acids, and L is sulfur, sulfoxide, oxygen or
methylene. Accordingly, either (i) aa.sup.1 is Adi and aa.sup.4 is
Glu or (ii) each of aa.sup.1 and aa.sup.4 is Adi. The compound (and
its conjugate) binds an SH2 domain in a protein comprising an SH2
domain, is non-phosphorylated, is redox-stable in vivo, and is
characterized by an IC.sub.50 in vivo of less than about 4.0 .mu.M
with respect to the SH2 domain in Grb2. Preferably, the compound
(and its conjugate) has an IC.sub.50 in vivo of less than or equal
to 2.0 .mu.M. Upon binding to the SH2 domain of Grb2, the compound
has a turn conformation. Optionally, one or more of Tyr.sup.3,
Val.sup.6, Met.sup.8 and Tyr.sup.9 is modified; desirably, the
modification does not result in a highly charged moiety that
prevents said compound from entering into a cell. Also, optionally,
there is a conservative or neutral amino acid substitution at
either one or both of Leu.sup.2 and Gly.sup.7.
[0025] Such compounds can be synthesized in accordance with methods
known in the art. Preferred methods are those that are set forth in
the Examples, such as Examples 1, 2 and 3. The compound of Example
1 has an IC.sub.50=0.64 .mu.M in the Biacore binding assay
described herein.
[0026] In addition to the above, the present invention provides a
conjugate comprising a compound as described above and a carrier
agent, which functions to internalize the compound into the cell.
Examples of carrier agents include a signal peptide, antennapedia
peptide, lipofectin and the like.
[0027] Such conjugates can be synthesized in accordance with
methods known in the art. A preferred method is set forth in
Example 12. The conjugate of Example 12 is an effective inhibitor
of protein association at 2 .mu.M concentration.
[0028] Also provided by the present invention is a method of
inhibiting binding of an SH2 domain in a protein comprising an SH2
domain to a target protein in an animal. The method comprises
contacting the protein comprising an SH2 domain with a target
protein-binding inhibiting amount of a compound or a conjugate as
described above. Upon binding of the SH2 domain by a compound or a
conjugate as described above, binding of a target protein is
inhibited. Preferably, the target protein is a growth factor
receptor, such as an EGFR. Alternatively and also preferably, the
target protein is erbB, in particular erbB-2. Other alternative and
preferred embodiments of the method include ones in which the
target protein is a morphology determining protein, such as FAK, a
cellular attachment protein, a proto-oncoprotein, an oncoprotein,
such as BCR-abl, or a mitogen-activated protein (MAP) kinase. In
one application of the method, preferably inhibition of binding of
a target protein by an SH2 domain in a protein comprising an SH2
domain prevents cancer, in particular breast cancer.
[0029] The method of inhibiting binding of a target protein by an
SH2 domain in a protein in an animal can further comprise
administering to the animal an anti-cancer agent, such as a cancer
chemotherapeutic agent, such as a cytotoxic agent or its prodrug,
radiation and/or a radioactive isotope. Examples of
anti-cancer/cytotoxic agents and their prodrugs include genistein,
okadaic acid, 1-.beta.-D-arabinofuranosyl-cytosine,
arabinofuranosyl-5-aza-cytosine, cisplatin, carboplatin,
angiostatin, endostatin, anti-Her-2/neu antibody, actinomycin D,
asparaginase, bis-chloro-ethyl-nitroso-urea, bleomycin,
chlorambucil, cyclohexyl-chloro-ethyl-nitroso-urea, cytosine
arabinoside, daunomycin, etoposide, hydroxyurea, melphalan,
mercaptopurine, mitomycin C, nitrogen mustard, procarbazine,
teniposide, thioguanine, thiotepa, vincristine, 5-fluorouracil,
5-fluorocytosine, adriamycin, cyclophosphamide, methotrexate,
vinblastine, doxorubicin, leucovorin, taxol, anti-estrogen agents
such as tamoxifen, intracellular antibodies against oncogenes, the
flavonol quercetin, Guan-mu-tong extract, retinoids such as
fenretinide, nontoxic retinoid analogues such as
N-(4-bydroxyphenyl)-retinamide (HPR), and monoterpenes such as
limonene, perillyl alcohol and sobrerol. Immunomodulators and
immunostimulants also can be used in the context of the present
inventive methods and include, but are not limited to, various
interleukins, cytokines, antibody preparations, and interferons.
Desirably, inhibition of binding of a target protein by an SH2
domain in a protein comprising an SH2 domain and administration of
an effective amount of an anti-cancer agent treats cancer.
[0030] In view of the above methods, the present invention further
provides a composition comprising (i) a compound or a conjugate as
described above and (ii) a carrier, such as a pharmaceutically
acceptable carrier. Pharmaceutically acceptable carriers are
well-known to those of ordinary skill in the art, as are suitable
methods of administration. The choice of carrier will be
determined, in part, by the particular method used to administer
the composition. One skilled in the art will also appreciate that
various routes of administering a composition are available, and,
although more than one route can be used for administration, a
particular route can provide a more immediate and more effective
reaction than another route. Accordingly, there are a wide variety
of suitable formulations of compositions that can be used in the
present inventive methods.
[0031] A compound or conjugate thereof as described above, alone or
in further combination with one or more other active agents, can be
made into a formulation suitable for parenteral administration,
preferably intraperitoneal administration. Such a formulation can
include aqueous and nonaqueous, isotonic sterile injection
solutions, which can contain antioxidants, buffers, bacteriostats,
and solutes that render the formulation isotonic with the blood of
the intended recipient, and aqueous and nonaqueous sterile
suspensions that can include suspending agents, solubilizers,
thickening agents, stabilizers, and preservatives. The formulations
can be presented in unit dose or multi-dose sealed containers, such
as ampules and vials, and can be stored in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile
liquid carrier, for example, water, for injections, immediately
prior to use.
[0032] Extemporaneously injectable solutions and suspensions can be
prepared from sterile powders, granules, and tablets, as described
herein.
[0033] A formulation suitable for oral administration can consist
of liquid solutions, such as an effective amount of the compound
dissolved in diluents, such as water, saline, or fruit juice;
capsules, sachets or tablets, each containing a predetermined
amount of the active ingredient, as solid or granules; solutions or
suspensions in an aqueous liquid; and oil-in-water emulsions or
water-in-oil emulsions. Tablet forms can include one or more of
lactose, mannitol, corn starch, potato starch, microcrystalline
cellulose, acacia, gelatin, colloidal silicon dioxide,
croscarmellose sodium, talc, magnesium stearate, stearic acid, and
other excipients, colorants, diluents, buffering agents, moistening
agents, preservatives, flavoring agents, and pharmacologically
compatible carriers.
[0034] Similarly, a formulation suitable for oral administration
can include lozenge forms, which can comprise the active ingredient
in a flavor, usually sucrose and acacia or tragacanth; pastilles
comprising the active ingredient in an inert base, such as gelatin
and glycerin, or sucrose and acacia; and mouthwashes comprising the
active ingredient in a suitable liquid carrier; as well as creams,
emulsions, gels, and the like containing, in addition to the active
ingredient, such carriers as are known in the art. An aerosol
formulation suitable for administration via inhalation also can be
made. The aerosol formulation can be placed into a pressurized
acceptable propellant, such as dichlorodifluoromethane, propane,
nitrogen, and the like.
[0035] A formulation suitable for topical application can be in the
form of creams, ointments, or lotions. A formulation for rectal
administration can be presented as a suppository with a suitable
base comprising, for example, cocoa butter or a salicylate. A
formulation suitable for vaginal administration can be presented as
a pessary, tampon, cream, gel, paste, foam, or spray formula
containing, in addition to the active ingredient, such carriers as
are known in the art to be appropriate.
[0036] The dose administered to an animal, such as a mammal,
particularly a human, in the context of the present invention
should be sufficient to effect a prophylactic or therapeutic
response as desired in the animal over a reasonable time frame. The
dose will be determined by the potency of the particular compound
or conjugate as described above, the severity of a condition being
treated, such as cancer, as well as the body weight and age of the
animal. The size of the dose also will be determined by the
existence of any adverse side effects that may accompany the use of
the particular compound or conjugate. It is always desirable,
whenever possible, to keep adverse side effects to a minimum.
[0037] The dosage can be in unit dosage form, such as a tablet or
capsule. The term "unit dosage form" as used herein refers to
physically discrete units suitable as unitary dosages for human and
animal subjects, each unit containing a predetermined quantity of a
compound or conjugate, alone or in combination with other active
agents, such as anticancer agents, calculated in an amount
sufficient to produce the desired effect in association with a
pharmaceutically acceptable diluent, carrier, or vehicle. The
specifications for the unit dosage forms of the present invention
depend on the particular embodiment employed and the effect to be
achieved, as well as the pharmacodynamics associated with each
compound or conjugate in the host animal. The dose administered
should be, with respect to a present inventive compound or
conjugate as set forth above, a "target protein binding-inhibiting
amount" or, with respect to an anti-cancer agent, an "effective
amount" or an amount necessary to achieve an "effective level" in
the animal. The "effective level" is used as the preferred endpoint
for dosing, the actual dose and schedule can vary, depending on
interindividual differences in pharmacokinetics, drug distribution,
and metabolism.
[0038] One skilled in the art can easily determine the appropriate
dose, schedule, and method of administration for the exact
formulation of the composition being used, in order to achieve the
desired "effective level" in the individual patient. One skilled in
the art also can readily determine and use an appropriate indicator
of the "effective level" of the compounds of the present invention
by a direct (e.g., tumor biopsy or radio-imaging of the tumor) or
indirect (e.g., PSA levels in the blood) analysis of appropriate
patient samples (e.g., blood and/or tissues).
[0039] A method of synthesizing a conjugate described above is also
provided. The method comprises:
[0040] (i) synthesizing from C-terminus to N-terminus a linear
side-chain protected peptide comprising from N-terminus to
C-terminus the amino acid sequence of the compound of claim 1 or 2
and the amino acid sequence of a carrier agent on an amide
resin,
[0041] (ii) N-terminally haloacetylating the peptide,
[0042] (iii) cleaving the peptide from the resin and, either
simultaneously or sequentially, deprotecting the side-chains of the
peptide,
[0043] (iv) nucleophilically displacing the N-terminal halo group
with the cysteine side-chain thiol functionality at from about pH 7
to about pH 8, and
[0044] (v) purifying the resulting conjugate. Preferably, the
haloacetylating is bromoacetylating or chloroacetylating.
[0045] The method is exemplified herein in Example 12. Suitable
side-chain protections are listed under "EXAMPLES" herein. The
resulting conjugate can be purified by any suitable means such as
reverse-phase HPLC.
EXAMPLES
[0046] The following examples serve to illustrate the present
invention and are not intended to limit the scope of the present
invention in any way. The PAL amide resin and
fluorenylmethoxycarbonyl (Fmoc) derivatives of standard amino acids
were obtained from Perkin-Elmer/Applied Biosystems Division (Foster
City, Calif.). Side-chain protections were as follows: Glu(t-Bu),
Tyr(t-Bu), Asn(Trt), and Cys(Trt).
Fmoc-.gamma.-carboxy-L-Glu(OtBu).sub.2-OH [Fmoc-Gla(OtBu).sub.2OH],
Fmoc-L-.alpha.-aminoadipic acid-.delta.-t-butyl ester
[Fmoc-Adi(OtBu)OH] and Fmoc-L-tyrosine(O-malonyl-di-OtBu)-OH
[Fmoc-Tyr(O-malonyl-di-OtBu)OH] were purchased from BACHEM
(Torrance, Calif.). Trifluoroacetic acid (TFA), triethylsilane
(TES) and chloroacetic acid were purchased from Fluka (Ronkonkoma,
N.Y.). O-benzotriazol-1-yl-N,N,N.sup.1,N.sup.1-tetrame- thyluronium
hexafluorophosphate/1-hydroxybenzotriazole (HBTU/HOBt) activation
of N.sup..alpha.-protected amino acids was employed for coupling
and 20% piperidine/dimethylformamide (DMF) was used for Fmoc
deprotection.
O-(7-azabenzotriazol-1-yl)-N,N,N.sup.1,N.sup.1-tetramethylu- ronium
hexafluorophosphate/1-hydroxy-7-azabenzotriazole/diisopropylethylam-
ine (HATU/HOAt/DIEA) in DMF was used for backbone cyclization;
7-azabenzotriazole-1-yl-oxy-tris(pyrrolidino)phosphonium-hexafluorophosph-
ate (PyAOP; available from Perspective Biosystems, Inc.,
Framington, Mass.)/HOAt/DIEA in DMF was used for backbone
cyclization of Adi-containing compounds. TFA/TES/H.sub.2O
(9.5:0.25:0.25) was used for the resin cleavage and side-chain
deblocking. The crude peptides were purified to homogeneity by
reverse-phase high-performance liquid chromatography (RP-HPLC).
HPLC conditions: Vydac C.sub.18 column (10.times.250 mm); solvent
gradient, A, 0.05% TFA in water; B, 0.05% TFA in 90% acetonitrile
in water with gradient indicated below; flow rate, 2.5 ml/min; UV
detector, 225 nm. Fast atom bombardment mass spectrometry (unit
resolution, glycerol matrix) was performed on a VG Analytical
7070E-HF mass spectrometer. The purity of products was
characterized by analytical HPLC and thin layer chromatography
(TLC). Amino acid analysis (6 N HCl, 110.degree. C., 24 h) was
carried out at the Protein Structure Laboratory (University of
California, Davis, Calif.).
Example 1
[0047] This example describes the synthesis of
cyclo(CH.sub.2CO-Gla.sup.1--
Leu.sup.2-Tyr.sup.3-Glu.sup.4-Asn.sup.5-Val.sup.6-Gly.sup.7-Met.sup.8-Tyr.-
sup.9-Cys)-amide [SEQ ID NO: 4; in SEQ ID NO: 1, L=S].
[0048] Using an ABI 433A peptide synthesizer and FastMoc chemistry
(Field et al., Pep. Res. 4: 95 (1991)), the linear side-chain
protected peptide Gla-LYENVGMYC [SEQ ID NO: 5] was synthesized on
the PAL amide resin (0.189 g, 0.1 mmol, 0.53 mmol/g) coupled with
the respective amino acids and the N.sup..alpha.-Fmoc group of the
resin-bound protected peptide was removed with 20% piperidine/DMF
(12 min). After cycles of deprotection and coupling,
NH.sub.2-Gla(OtBu).sub.2-Leu-Tyr(tBu)-Glu(OtBu)-Asn(Trt)-Va-
l-Gly-Met-Tyr(tBu)-Cys(Trt)-C(O)NH-Resin [SEQ ID NO: 6] was
obtained. The resin-bound protected peptide was N-terminally
chloroacetylated with (ClCH.sub.2CO).sub.2O. For this purpose,
(ClCH.sub.2CO).sub.2O was prepared by mixing 0.5 M
ClCH.sub.2COOH/DCM (48 mg, 0.5 mmol, 1.0 ml) and 0.5 M DCC/DCM (52
mg, 0.25 mmol, 0.5 ml) for 0.5 h at RT. The precipitated
dicyclohexylurea (DCU) was filtered off and the filtrate was added
to the peptide resin. The resulting reaction mixture was shaken at
RT for 6 h; Ninhydrin test was negative. The peptide
ClCH.sub.2C(O)-NH-Gla-Leu-Tyr-Glu-Asn-Val-Gly-Met-Tyr-C(CH.sub.2SH)C(O)NH-
.sub.2 [SEQ ID NO: 7] was cleaved from the resin using TFA
containing 2.5% each (v/v) of TES and deionized H.sub.2O (2 h).
Two-thirds of the cleavage reagent mixture was evaporated under
N.sub.2 and the mixture was triturated in ice-cold ether to isolate
the product. The precipitated crude peptide,
ClCH.sub.2CO-Gla-Leu-Tyr-Glu-Asn-Val-Gly-Met-Tyr-Cys-amide [SEQ ID
NO: 7], was dissolved in 50 ml water, and added dropwise into 100
ml of H.sub.2O solution, which was adjusted to pH 8-9 with
triethylamine, repeatedly. Under basic conditions, the
N-chloroacetylated linear peptide spontaneously cyclized by
intramolecular nucleophilic displacement of the chloro group by
cysteine thiol. Cyclization was monitored by HPLC. After 6-7 h at
RT, the solution was acidified with 30% AcOH aqueous solution and
lyophilized. The product was purified by RP-HPLC (gradient 20-70% B
over 27 min, Rt 11.4 min) to provide
cyclo(CH.sub.2CO-Gla.sup.1-Leu.sup.2-
-Tyr.sup.3-Glu.sup.4-Asn.sup.5-Val.sup.6-Gly.sup.7-Met.sup.8-Tyr.sup.9-Cys-
)-amide [SEQ ID NO: 4] in 30% yield. FAB-MS (M+H)+1303.4 (calc'd
1303.5). Amino acid analysis: Asp+S-CM-Cys 1.51 (1 each), Val 1.00
(1), Leu 1.23 (1), Glu+Gla 1.88 (1 each), Gly 1.18 (1), Tyr 1.83
(2), Met 0.85 (1).
Example 2
[0049] This example describes the synthesis of
cyclo(CH.sub.2CO-Adi.sup.1--
Leu.sup.2-Tyr-Glu.sup.4-Asn.sup.5-Val.sup.6-Gly.sup.7-Met.sup.8-Tyr.sup.9--
Cys)-amide [SEQ ID NO: 8].
[0050]
Cyclo(CH.sub.2CO-Adi.sup.1-Leu.sup.2-Tyr.sup.3-Glu.sup.4-Asn.sup.5--
Val.sup.6-Gly.sup.7-Met.sup.8-Tyr.sup.9-Cys)-amide [SEQ ID NO: 8]
was prepared analogously to
cyclo(CH.sub.2CO-Gla.sup.1-Leu.sup.2-Tyr.sup.3-Gl-
u.sup.4-Asn.sup.5-Val.sup.6-Gly.sup.7-Met.sup.8-Tyr.sup.9-Cys)-amide
[SEQ ID NO: 4] as described in Example 1. Product characterization:
RP-HPLC R.sub.t=13.6 min (gradient 20-70% B over 27 min) in overall
yield of 40%. FAB-MS (M+H).sup.+1273.4 (calc'd 1273.5). Amino acid
analysis: Asp 0.43 (1), S-CM-Cys 0.98 (1), Adi 0.97 (1), Val 1.00
(1), Leu 1.21 (1), Glu 1.09 (1), Gly 1.13 (1), Tyr 1.87 (2), Met
0.93 (1).
Example 3
[0051] This example describes the synthesis of
cyclo(CH.sub.2CO-Adi.sup.1--
Leu.sup.2-Tyr.sup.3-Adi.sup.4-Asn.sup.5-Val.sup.6-Gly.sup.7-Met.sup.8-Tyr.-
sup.9-Cys)-amide [SEQ ID NO: 9].
[0052]
Cyclo(CH.sub.2CO-Adi.sup.1-Leu.sup.2-Tyr.sup.3-Adi.sup.4-Asn.sup.5--
Val.sup.6-Gly.sup.7-Met.sup.8-Tyr.sup.9-Cys)-amide [SEQ ID NO: 9]
was prepared analogously to
cyclo(CH.sub.2CO-Gla.sup.1-Leu.sup.2-Tyr.sup.3-Gl-
u.sup.4-Asn.sup.5-Val.sup.6-Gly.sup.7-Met.sup.8-Tyr.sup.9-Cys)-amide
[SEQ ID NO: 4] as described in Example 1. Product characterization:
RP-HPLC R.sub.t=13.8 min (gradient 20-70% B over 27 min). FAB-MS
(M+H)+1287.2 (calc'd 1287.5).
Example 4
[0053] This example describes the synthesis of an L=sulfoxide
linkage in a compound of any of Examples 1-3, in which the
Met.sup.8 is simultaneously substituted by the oxidation-stable
norleucine (Nle) amino acid. For purposes of illustration, the
synthesis of cyclo(CH.sub.2CO-Glu.sup.1-Leu-
.sup.2-Tyr.sup.3-Glu.sup.4-Asn.sup.5-Val.sup.6-Gly.sup.7-Nle.sup.8-Tyr.sup-
.9-Cys)-amide-sulfoxide [SEQ ID NO: 10] is described.
[0054] Thus, for example, the resin-bound, protected peptide
NH.sub.2-Glu(OtBu)-Leu-Tyr(OtBu)-Glu(OtBu)-Asn(Trt)-Val-Gly-Met-Tyr(OtBu)-
-Nle-C(O)NH-resin [SEQ ID NO: 11] was synthesized using FastMoc
chemistry. This resin-bound peptide was N-terminally
chloroacetylated, cleaved from the resin, side-chain deprotected
and thioether cyclized according to the above-described procedures
to produce cyclo(CH.sub.2CO-Glu.sup.1-Leu.sup.-
2-Tyr.sup.3-Glu.sup.4-Asn.sup.5-Val.sup.6-Gly.sup.7-Nle.sup.8-Tyr.sup.9-Cy-
s)-amide [SEQ ID NO: 12]. The resulting thioether was oxidized by
5%H 202 aqueous solution (1 mg/ml) with stirring at room
temperature for 6 hr. The oxidation process was monitored by HPLC.
After lyophilizing, the crude product was purified by RP-HPLC and
two diastereoisomers were obtained with Rt of 13.0 min and 14.2 min
(gradient 25-40% B over 30 min). The faster-eluting fraction:
FAB-MS (M+H).sup.+1258.1 (calcd 1257.5). Amino acid analysis: Asp
0.90(1), Val 1.03 (1), Leu 1.09 (2), Glu 1.79 (2), Gly 0.93 (1),
Tyr 1.12 (2)*, Nle 1.04 (1). The slower-eluting fraction: FAB-MS
(M+H).sup.+1258.1 (calcd 1257.5). Amino acid analysis: Asp 0.79
(1), Val 1.03 (1), Leu 1.12 (1), Glu 1.64 (2), Gly 1.01 (1), Tyr
(0.85) (2)*, Nle 1.08 (1).
Example 5
[0055] This example describes the synthesis of an L=methylene
linkage in a compound of any of Examples 1-3. For purposes of
illustrate, the synthesis of
cyclo(Glu.sup.1-Leu.sup.2-Tyr.sup.3-Glu.sup.4-Asn.sup.5-Val.-
sup.6-Gly.sup.7-Nle.sup.8-Tyr.sup.9-[L-.alpha.-aminoadipate])-amide
[SEQ ID NO: 13] is described. (NOTE: In this structure, the
side-chain carboxyl of .alpha.-aminoadipic acid was cyclized by
forming an amide bond with the N-terminal amino functionality of
Glu.sup.1. Also, the unnatural amino acid
N.sup..alpha.-Fmoc-L-.alpha.-aminoadipic acid-.delta.-allyl ester
was incorporated onto the synthesis resin as the first amino
acid.)
[0056] The linear peptide
NH.sub.2-Glu(OtBu)-Leu-Tyr(OtBu)-Glu(OtBu)-Asn(T-
rt)-Val-Gly-Met-Tyr(OtBu)-Adi(OAI)-NH-resin [SEQ ID NO: 14] was
assembled by standard solid phase methods (using the ABI 433A
Peptide Synthesizer and FastMoc chemistry). Next, the allyl group
was removed from the Adi sidechain. Accordingly, 0.292 g of the
peptide resin (0.083 mmol) was suspended on 10 ml of DMF-THF-0.5 M
HCl-morpholine (2:2:1:0.1 v/v) and treated with Pd(PPh.sub.3).sub.4
(0.593 g, 0.513 mmol) for 2.5 hr under N.sub.2. The resin was then
washed with THF (3.times.2 min), DMF (3.times.2 min), DCM
(3.times.2 min), DIEA-DCM (1:19) (3.times.2 min), DCM (3.times.2
min), sodium diethyldithiocarbamate-DMF (5 g/l; 3.times.15 min),
DMF (5.times.2 min), and DCM (3.times.2 min). The head-to-tail
side-chain cyclization of the resin-bound peptide was performed
with PyAOP (458 mg, 0.88 mmol), HOAt (120 mg, 0.88 mmol), and DIEA
(306 ml, 1.76 mmol) in dry DMF at room temperature overnight. After
resin cleavage and side-chain deprotection with TFA-TES-H.sub.2O
(9.5:0.25:0.25, v/v), the crude peptide was purified by RF-HPLC,
R.sub.t=13.5 min (gradient 20-70% B over 30 min).
[0057] FAB-MS (M+H)+1241.2 (calcd 1241.5).
Example 6
[0058] This example describes a potential method of synthesis of an
L=oxygen linkage in a compound of any of Examples 1-3.
[0059] An ether-cyclized compound of any of Examples 1-3 is
prepared in accordance with the method of Example 5, except that
the N.sup..alpha.-Fmoc-L-.alpha.-aminoadipic acid-6-allyl ester is
replaced with N.sup..alpha.-Fmoc-L-(O-carboxymethyl-allyl
ester)-serine.
Example 7
[0060] This example describes the affinity characteristics of the
present inventive compounds for binding to the SH2 domain of
Grb2.
[0061] The competitive binding affinity of the present inventive
compounds was 30 assessed using Biacore Surface Plasmon Resonance
(SPR) methods utilizing a Biacore 2000 instrument. The
concentrations at which half-maximal competition was observed
(IC.sub.50) were determined by mixing the inhibitor with
recombinant GST-Grb2 SH2 protein and measuring the amount of
competitive binding at equilibrium to an immobilized (via biotin
conjugation to streptavidin coated surface of the Biacore sensor
chip SA; 2 nM concentration) SHC(pTyr-317)-9-mer phosphopeptide in
a manner similar to that reported previously (Oligino et al.
(1997), supra). Specifically, competitive binding assays were
carried out at 5 .mu.l/min flow rate, with GST-Grb2-SH2 (150-250
nM) and a cyclic peptide inhibitor (10 nM-2 mM) in
phosphate-buffered saline (PBS), pH 7.4, containing 0.01% P-20
surfactant (Biacore, Inc., Piscataway, N.J.) for a total of 10 min.
The change in response units (RU) at equilibrium was measured from
the absorption profiles and the percent of control (no added
peptide) was calculated. The IC.sub.50 for
cyclo(CH.sub.2CO-Adi.sup-
.1-Leu.sup.2-Tyr.sup.3-Glu.sup.4-Asn.sup.5-Val.sup.6-Gly.sup.7-Met.sup.8-T-
yr.sup.9-Cys)-amide [SEQ ID NO: 8] was 3.45.+-.0.15 .mu.M, whereas
the IC.sub.50's for
cyclo(CH.sub.2CO-Adi.sup.1-Leu.sup.2-Tyr.sup.3-Adi.sup.4--
Asn.sup.5-Val.sup.6-Gly.sup.7-Met.sup.8-Tyr.sup.9-Cys)-amide [SEQ
ID NO: 9] and
cyclo(CH.sub.2CO-Gla.sup.1-Leu.sup.2-Tyr.sup.3-Glu.sup.4-Asn.sup.5-
-Val.sup.6-Gly.sup.7-Met.sup.8-Tyr.sup.9-Cys)-amide [SEQ ID NO: 4]
were 3.05.+-.0.05 .mu.M and 0.64.+-.0.10 .mu.M, respectively. The
results represent the mean value of at least two independent
experiments.
Example 8
[0062] This example describes the ability of the present inventive
compounds to inhibit growth factor receptor (p185.sup.erbB-2)
interaction with Grb2 in cell homogenates.
[0063] Cell lysates were prepared from the serum-treated
erbB-2-over-expressing breast cancer cell line MDA-MB-453 using 1%
Triton X-100 in PBS containing 0.2 mM NaVO.sub.4. Lysates were
incubated with the present inventive cyclic peptide inhibitors
specified in Example 1 and Example 12, at concentrations of 0.1,
0.4, 2, 10 and 50 .mu.M. No inhibitor was used as a control. Cell
lysates were prepared using 1% Triton X-100 in PBS containing 0.2
mM NaVO.sub.4. Grb2 and associated Grb2-binding proteins were
immunoprecipitated from each lysate (500 .mu.g) with anti-Grb2
antibodies and collected using protein A Sepharose using methods
previously described. Immunoprecipitated proteins were separated by
SDS-PAGE on 8-16% gradient gels (Novex). pTyr-containing proteins
were detected by Western blotting using anti-phosphotyrosine
antibodies (Upstate Biotechnology Inc.). Previous experiments have
shown that a major tyrosine phosphorylated protein in MDA-MB-453
cells is p185.sup.erbB-2, which is overexpressed as a consequence
of gene amplification. The same membrane was then re-probed with
anti-Grb2 antibody for Western blotting to demonstrate equal
loading. A dose-dependent inhibition of the p185.sup.erbB-2
receptor association with the Grb2 adapter protein was observed. A
half-maximal inhibition of this protein association was observed at
2 .mu.M with the inhibitors of Examples 1 and 12.
Example 9
[0064] This example describes the ability of the present inventive
compounds to inhibit intracellular protein-protein interaction
between p 185.sup.erbB-2 and Grb2.
[0065] The compound of Example 1, and the conjugate of Example 12,
were tested in MDA-MB-453 cells (breast cancer cells that
overexpress p185.sup.erbB-2 receptor) at inhibitor concentrations
of 0.1, 0.4, 2, 10 and 50 .mu.M. In the control experiment, no
inhibitor was used. The cells were treated for 3 hrs in serum-free
IMEM medium. The cells were washed twice with ice-cold PBS to
remove the residual inhibitory compound that was not
internalized.
[0066] Cell lysates were prepared using 1% Triton X-100 in PBS
containing 0.2 mM NaVO.sub.4. Grb2 and associated Grb2-binding
proteins were immunoprecipitated from each lysate (500 .mu.g) with
anti-Grb2 antibodies and collected using protein A Sepharose using
methods previously described. Immunoprecipitated proteins were
separated by SDS-PAGE on 8-16% gradient gels (Novex).
pTyr-containing proteins were detected by Western blotting using
anti-phosphotyrosine antibodies (Upstate Biotechnology Inc.).
Previous experiments have shown that a major tyrosine
phosphorylated protein in MDA-MB-453 cells is p185.sup.erbB-2,
which is overexpressed as a consequence of gene amplification. The
same membrane was then re-probed with anti-Grb2 antibody for
Western blotting to demonstrate equal loading.
[0067] In these whole cell assays the agents were applied to the
cell culture media of MDA-MB-453 cells. These conditions required
that compounds transit through the cell membrane. A clear
dose-dependent reduction in the associated p185.sup.erbB-2 bound to
Grb2 was found in the cells when cells were pre-treated with
increasing amounts of the peptide conjugated to carrier (Example
12). This carrier-conjugated peptide effectively inhibited the
protein association at 2 .mu.M. Moreover, the peptide (Example 1)
that was not conjugated to a carrier also showed effective
inhibition of p185.sup.erbB-2/ Grb2 association in cells at 50
.mu.M concentration, indicating an ability to cross the cell
membrane.
Example 10
[0068] This example describes the ability of the present inventive
compounds to inhibit MAP kinase activation.
[0069] Serum-starved SAA cells are treated with a compound of any
of Examples 1-3 and EGF. Cells are washed and lysed and a fraction
of cell lysate is immunoprecipitated with a polyclonal anti-ERK2
antibody (Santa Cruz). The complexes on protein A Sepharose beads
are washed and incubated with 10 .mu.Ci of .gamma.-.sup.32P-ATP
(ICN Biochemicals, Cleveland, Ohio) and 0.25 mg/ml of myelin basic
protein (Sigma) in 50 mM HEPES (pH 7.2), 10 mM magnesium acetate, 1
mM dithiothreitol, and 20 mM ATP at 30.degree. C. for 20 min. The
kinase reaction is quenched with sample buffer. The samples are
separated on 15% SDS-PAGE, transferred to nitrocellulose and
subjected to autoradiography.
Example 11
[0070] This example describes the application of molecular modeling
for predicting the binding mode of the present inventive compounds
to the SH2 domain of Grb2.
[0071] Molecular modeling simulations were performed with the
Insight II 97/Discover 3.0 modeling package from Molecular
Simulations, Inc., San Diego, Calif., with the cff91-force field.
The X-ray structure of the KPFpYVNV [SEQ ID NO: 15] peptide ligand
bound to the Grb2-SH2 was used as a starting geometry (Rahuel et
al., Nature Struct. Biology 3: 586 (1996)). The positions of the
backbone atoms and the side-chain atoms of those residues in the
turn region (FpYVNV [SEQ ID NO: 16]) of the reference peptide
sequence were used as the initial atom positions of the
sub-sequence LYENV [SEQ ID NO: 17], since a turn structure has been
predicted for this sub-sequence also. Then, the remaining residues
of a compound in accordance with the present invention were added
to the model. The --CO(Y), E, N and NH(V)- atoms, which comprise
the turn, and the protein atoms were kept fixed during the
following minimization and 50 simulated annealing (SA) situation.
In each SA run, a different random seed was used. The same
minimized starting geometry was subjected to a molecular dynamics
simulation at 2,000 K for 10 ps and consecutively cooled in 5 K
increments to 5 K during 195 ps. The final structure with the
lowest energy was taken.
[0072] Adi.sup.1 or Gla.sup.1 remarkably improved the binding
efficacy over Glu.sup.1 by 6-fold and 31-fold, respectively. The
side chains of Adi.sup.1 and Gla.sup.1 are believed to have similar
and overlapping roles with the phosphorylated Tyr.sup.3 in the
naturally occurring ligand. Based on such results, it is believed
that amino acids in the first position of the compound that possess
flexible alkyl chains can orient the polar carboxyl groups to an
optimal position for bonding interactions with the SH2 domain of a
target protein.
[0073] Docking of
cyclo(CH.sub.2CO-Gla.sup.1-Leu.sup.2-Tyr.sup.3-Glu.sup.4-
-Asn.sup.5-Val.sup.6-Gly.sup.7-Met.sup.8-Tyr.sup.9-Cys)-amide [SEQ
ID NO: 4] into the Grb2-SH2 binding pocket, assuming that Tyr.sup.3
and Asn.sup.5 occupy near proximal binding sites before dynamic
simulation, indicates that Gla.sup.1 is favored over Glu.sup.1
because the second carboxyl group of the side-chain can undergo
additional interactions within the binding pocket of pTyr of the
naturally occurring ligand. The resulting modified carboxyl
side-chain(s) provide for more favorable ionic interactions with
the guanidino functionalities of Arg 67 and Arg 86 in Grb2.
Example 12
[0074] This example describes the synthesis and characterization of
an active peptide with high affinity for Grb2 conjugated to a
signal peptide as a carrier agent.
[0075] Using an ABI 433A peptide synthesizer and FastMoc chemistry,
the linear side-chain protected peptide,
Gla.sup.1-Leu.sup.2-Tyr.sup.3-Glu.su-
p.4-Asn.sup.5-Val.sup.6-Glu.sup.7-Met.sup.8-Tyr.sup.9-Cys-Ala-Ala-Val-Ala--
Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro [SEQ ID NO: 18],
was synthesized on a PAL amide resin, which comprises the signal
peptide (in italics) of the Kaposi fibroblast growth factor (Rojas
et al., J. Biol. Chem. 271: 27456-27461 (1996)). The resin-bound
peptide was N-terminally chloroacetylated, cleaved from the resin
and side-chain deprotected, using similar conditions to those
described in Example 1. Cyclization was achieved by nucleophilic
displacement of the N-terminal chloride with the cysteine
side-chain thiol functionality at pH 8 over a period of 6 hours.
The resulting peptide
cyclo(CH.sub.2CO-Gla.sup.1-Leu.sup.2-Tyr.sup.3-Glu.-
sup.4-Asn.sup.5-Val.sup.6-Gly.sup.7-Met.sup.8-Tyr.sup.9-Cys)-NH-Ala-Ala-Va-
l-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro [SEQ ID NO:
19] was purified by RP-HPLC. HPLC conditions: Vydac C4 column
(20.times.250 mm), flow rate 10 ml/min, solvent A, 0.05% TFA in
water; B, 0.05% TFA in 90% acetonitrile in water; UV detector, 225
nm; gradient 40-90% B over 27 in; R.sub.t=20.6 min. FAB-MS
(M+Na).sup.+2823.4 (calc'd 2824.3).
[0076] The references, including journal articles, patent
applications and patents, cited herein are hereby incorporated in
their entireties by reference.
[0077] While this invention has been described with an emphasis
upon preferred embodiments, it will be obvious to those of ordinary
skill in the art that variations of the preferred compounds,
conjugates, compositions and methods may be used and that it is
intended that the invention may be practiced otherwise than as
specifically described herein. Accordingly, this invention includes
all modifications encompassed within the spirit and scope of the
invention as defined by the following claims.
* * * * *